DNA Replication & Repair Chapter 16 Pages 292-301 Cell Cycle http://scientopia.org/img-archive/scicurious/img_862.png Models of DNA replication Fig. 16.8 Models of DNA replication Conservative model Semiconservative model Dispersive model Daughter duplex Daughter duplexes are Daughter duplexes are made of 2 newly made up of one made up of segments synthesized strands. parental strand and one of parental DNA and Parent duplex newly synthesized newly synthesized conserved. strand DNA Matthew Meselson & Franklin Stahl (1958)Performed an experiment to determine which model of DNA replication was true See first half of video up to CsCl centrifugation: http://highered.mcgraw-hill.com/olc/dl/120076/bio22.swf http://www.pnas.org/content/101/52/17889/F1.medium.gif Meselson & Stahl Experiment Step 1 DNA contains nitrogen atoms (bases) First grew E. Coli cells in heavy nitrogen (15N) When cells are centrifuged the DNA will show up heavy Density Gradient Centrifugation Density Gradient Centrifugation Meselson & Stahl Experiment Step 2 Then transferred E. Coli cells into a medium with light nitrogen (14N) Cells were allowed one round of replication Then they were centrifuged Meselson & Stahl Experiment Step 2 ■ Predict what the centrifuge tube would show with each Conservative model Semi- conservative Dispersive Meselson & Stahl Experiment Step 2 ■ Which one did he see? Conservative Semi- conservative Dispersive Meselson & Stahl Experiment Step 3 Cells were then allowed to grow for a second round of replication in the light medium (14N) And they were once again centrifuged Meselson & Stahl Experiment Step 3 ■ Predict what the centrifuge tube would show with each Conservative model Semi- conservative Dispersive Meselson & Stahl Experiment Step 3 ■ Which one did he see? Conservative Semi- conservative Dispersive Meselson & Stahl Experiment Summary 15N 14N 14N Meselson & Stahl Experiment Summary Start with 15N Add 14N Add 14N Round of Conservative Semiconservative Dispersive replication model model model Two bands First One band One band medium: 15N14N replication Heavy: 15N15N medium: 15N14N Light: 14N14N Two bands Two bands One band at a weight that Second Medium: 15N14N is in between the medium replication Heavy: 15N15N Light: 14N14N and light band weights Light: 14N14N Meselson & Stahl Experiment Summary Model Conservative Semi- Dispersive conservative Generation I Generation II Meselson & Stahl Experiment Basic concept of DNA replication (video) http://www.hhmi.org/biointeractive/dna/DNAi_replication_schematic.html Basic concept of DNA replication During DNA replication, base pairing enables existing DNA strands to serve as templates for new complimentary strands Fig. 16.7 2. DNA Replication Mechanism Initiation Elongation Termination DNA replication: Initiation Origins of replication (ori): Special sites on DNA where replication begins Replication Initiation: Prokaryote Replication begins at one fixed origin (only 1 ori) Replication proceeds bidirectionally until the DNA is replicated Replication Initation: Eukaryote More than one origin of replication (thousands of origin sites per chromosome) Replication forks and bubbles At the origin sites, the DNA strands separate forming a replication “bubble” with replication forks at each end. Fig. 16.10 Replication forks and bubbles The replication bubbles elongate as the DNA is replicated and eventually fuses. Fig. 16.10 Proteins in Replication Initiation Sticky Tack Demo to show action of helicase, SSBP and topoisomerase Yarn Sticky tack or tape scissors ■ Watch first half of video (before the fork): http://highered.mcgraw-hill.com/olc/dl/120076/bio23.swf Proteins in Replication Initiation http://eprots.pdbj.org/eprots/index_en.cgi?PDB%3A3BEP Proteins in Replication Initiation ■ Helicase: enzyme that disrupts H bonds between two strands of DNA to separate the template DNA strands at the replication fork. ■ Single-strand binding proteins (SSBPs): proteins that bind to unwound single-stranded regions of DNA to keep the template strands apart during replication ■ Topoisomerases: enzymes that can break bonds in DNA and then reforms the bonds ■ Purpose is to release the twists in DNA that are generated during DNA replication ■ Example of a topoisomerase: DNA gyrase Priming DNA for replication ■ Blue line: DNA to be copied ■ Pink line: RNA nucleotides added = Primer ■ Light pink blob: Enzyme that adds RNA = RNA polymerase Fig. 16.13 Priming DNA for replication Primer: a short segment of RNA needed to initiate DNA replication Note: all nucleic acids are formed in the 5’ to 3’ direction, even RNA (thus primers) Primase: an RNA polymerase (RNAP) which synthesizes the primer by adding ribonucleotides that are complementary to the DNA template Polymerase: enzyme that makes polymers Why is priming required? Fig. 16.13 Why is priming required? Due to the different abilities of RNA polymerase (RNAP) versus DNA polymerase (DNAP) RNAP: can start a new chain without an existing end All it needs is a template E.g. primase DNAP: can only add nucleotides to the end of an existing chain can never start a new chain because it needs the 3’ OH DNA Polymerase (DNAP) Enzyme which synthesizes nucleotide chains Prokaryotes: DNA polymerase I, II, III, IV & V Eukaryotes: over 15 different types named with Greek letters (e.g. DNAP ) DNA Replication: Elongation Fig. 16.11 DNA Replication: Elongation DNA polymerase III (DNAP III) catalyzes the elongation of DNA molecules by adding nucleotides to the 3’ end of a pre-existing nucleotide As each nucleotide is added, the last two phosphate groups are hydrolyzed to form pyrophosphate. Pyrophosphate is broken down into two phosphates NTP NMP + 2P DNA Elongation DNA Polymerase DNA polymerase I (DNAP I) replaces the RNA primer with DNA complementary to the template DNA Elongation DNAP III: elongates DNA strand DNAP I: replaces DNA polymerase III RNA with DNA DNA polymerase I Fig. 16.14 The problem at the fork Due to antiparallel nature of DNA One parental strand has its 3’ end at the fork while the other parental strand has its 5’ end at the fork. But DNA synthesis can only proceed in a 5’ 3’ direction. Directionality A strand of DNA can only add nucleotides onto its 3’ end DNA elongation only proceeds in the 5’ to 3’ direction DNAP must move along the template strand’s 3’ to 5’ direction. The leading and lagging strands Leading strand: is synthesized continuously Lagging strand: is synthesized in short, discontinuous segments of 1000-2000 nucleotides called Okazaki fragments Replication Fork http://kvhs.nbed.nb.ca/gallant/biology/replication_overview.jpg Fig. 16.16 Lagging strand: Okazaki fragments DNAP III synthesizes the DNA DNAP I replaces the RNA primer with DNA complementary to the template DNA ligase joins broken pieces of DNA by catalyzing the formation of phosphodiester bonds DNA Ligase http://fhs-bio-wiki.pbworks.com/f/DNA%20Ligase%20reaction.jpg Animations: Replication at the Fork, Leading & Lagging Second half of video:Strandshttp://highered.mcgraw- hill.com/olc/dl/120076/bio23.swf Video: http://highered.mcgraw- hill.com/olc/dl/120076/micro04.swf DNA Replication Machinery III III I I Fig. 16.15 The DNA Replication Complex Proteins involved in DNA replication form a single large complex anchored to the fibers in the nucleus. Stationary complex of DNA polymerase molecules pull in the parental DNA and produce newly made daughter DNA molecules. DNA Replication Components http://www.nature.com/nature/journal/v421/n6921/images/nature01407-i1.0.jpg Video: 3D model visualization and explanation of replication http://www.hhmi.org/biointeractive/dna/DNAi_replication_vo2.html DNA replication: Termination ■ DNA replication ends when: ■ Reach the end of the chromosome ■ Replication bubble / fork meets another replication bubble/ fork DNA Replication Tutorials http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/dn a-rna2.swf http://www.wiley.com/legacy/college/boyer/0470003790/animation s/replication/replication.swf http://www.wiley.com/college/pratt/0471393878/student/animation s/dna_replication/index.html http://www.mcb.harvard.edu/Losick/images/TromboneFINALd.swf http://www.johnkyrk.com/DNAreplication.html HW Questions Write out the point form summary of the steps in DNA replication. Include all the enzymes in the correct order. Since DNAP can only polymerize off an available 3’ end, where does DNAP I (the one that removes the RNA primer and replaces it with DNA) get that 3’ end from? Compare prokaryotic and eukaryotic replication and repair mechanisms by describing the differences. Use a comparative chart if possible. 3. DNA Repair: Proofreading Error Rate Types of Errors Exonuclease Endonuclease Proofreading DNA Error rate Average human chromosome has 150,000,000 bp Initial pairing error: 1 in 10,000 bp = 15,000 errors per replication… that’s a LOT Final error: 1 in 1,000,000,000 bp (1 in 109) Mechanisms in place to proofread errors as DNA is being replicated (exonuclease) Cell also continuously monitors and repairs DNA outside of replication (endonuclease) Types of Errors in DNA Mismatch mutations: incorrectly paired bases one of the most common types of errors during replication Missing bases Fused bases UV light A common cause of DNA damage Produces pyrimidine dimers (a type of fused base) http://www.nature.com/scitable/nated/content/19185/pierce_17_24_FULL.jpg Xeroderma Pigmentosum (XP) A condition where individual is unable to repair damage caused by UV light Individuals may need to avoid sunlight completely (“children of the night”) Leads to early skin cancer